During embryogenesis, the proliferation of glioblasts and their differentiation into several glial cell types appear to be closely coordinated with the development of neurons. During early postnatal development of the brain, glial cell proliferation continues at significant levels after neuronal proliferation has mostly ceased. In general, glial cell proliferation in the brain remains strongly inhibited throughout adult life, except in tumorigenesis and after injury.
The control of glial cell proliferation is an important factor in the regulation of neural development, regeneration, and tumorigenesis. The experiments described herein provide evidence for a new functional role of N-CAM in the control of glial cell proliferation in vitro and in vivo.
After physical injury to the central nervous system (CNS), astrocytes respond by changes in cell morphology, increased expression of glial fibrillary acidic protein (GFAP), hypertrophy, and rapid proliferation (Ludwin, S. K. (1984) Nature 308, 274-275). Such reactive astrocytes form glial scars in a process of gliosis that appears to interfere with neuronal regeneration in the adult CNS (Barrett, C. P. et al. (1984) Exp. Neurol. 84, 374-385; Reier, P. J. & Houle, J. D. (1988) In Advances in Neurology, (Waxman, S. G., ed.). Raven Press, New York, pp. 87-138).
Glial cell proliferation can be influenced by a variety of factors. Stimulatory effects are exerted by various growth factors (Kniss, D. A. & Burry, R. W. (1988) Brain Res. 439, 281-288), interleukin-6 (IL-6) (Selmaj, K. W. et al. (1990) J. Immunol. 144, 129-135), interleukin-1.beta. (IL-1.beta.) (Giulian, D. & Lachman, L. B. (1985) Science 228, 497-499), tumor necrosis factor (Barna, B. P. et al. (1990) J. Neuroimmunol. 30, 239-243) and .gamma.-interferon (Yong, V. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 7016-7020). The mitogenic effects of several of these factors can be inhibited by transforming growth factor-.beta. (Hunter, K. E., Sporn, M. B. & Davies, A. M. (1993) Glia 7, 203-211) which is produced by neurons (Flanders, K. C. et al. (1991) Development 113, 183-191).
Further evidence points to a direct involvement of neurons in the control of glial cell proliferation. The addition of purified cerebellar granule neurons to cerebellar astrocytes (Hatten, M. E. (1987) J. Cell Biol. 104, 1353-1360) or of hippocampal neurons to glia (Gasser, U. E. & Hatten, M. E. (1990) J. Neurosci. 10, 1276-1285) resulted in a decrease in glial cell proliferation. This inhibition depends on a proper ratio of neurons to glia of about 4 to 1, ensuring that astrocytes make surface contact with the added neurons. A cell membrane preparation of cerebellar granule neurons also inhibited proliferation, while medium conditioned by such neurons did not, indicating that membrane-associated molecules might be involved in the neuronal control of glial cell proliferation.
N-CAM (Edelman, G. M. (1986) Ann. Rev. Cell Biol. 2, 81-116; Cunningham, B. A. et al. (1987) Science 236, 799-806) has an important regulatory role in the developing nervous system and is present in adult nervous tissue. While the traditional view centers in the role of N-CAM in providing adhesion to the substrate and other cells, there is evidence that N-CAM can mediate signalling across the cell membrane in N-CAM mediated neurite outgrowth (Doherty, P. & Walsh, F. S. (1994) Current Opinion in Neurobiology 4, 49-55).
Astrocytes express a variety of adhesion molecules, among them the cell adhesion molecules N-CAM, and L1, and the extracellular matrix protein laminin. N-CAM has been found in astrocytes in vivo (Bartsch, U. et al. (1989) J. Comp. Neurol. 284, 451-462) as well as in primary astrocyte cultures (Noble, M. et al. (1985) Nature (London) 316, 725-728) although normal levels of N-CAM in glia are low in comparison to those in neurons (Nybroe, O. et al. (1985) J. Cell. Biol. 101, 2310-2315). The amount of N-CAM expressed on the surface of astrocytes (Smith, G. M. et al. (1993) J. Neurochem. 60, 1453-1466) as well as in the nervous system as a whole (Chuong, C.-M. & Edelman, G. M. (1984) J. Neurosci. 4, 2354-2368; Linnemann, D. et al. (1993) Int. J. Devl. Neurosci. 11, 71-81) decreases significantly as the CNS matures as compared to the amounts seen in prenatal and early postnatal development.
Levels of N-CAM have been shown to change after injury to nervous tissue. For example, after neurotoxin-induced brain damage, glial cells re-express high levels of the highly sialylated embryonic form of N-CAM (Le Gal La Salle, G. et al. (1992) J. Neurosci. 12, 872-882). Peripheral nerve injury leads to the expression of high levels of sialic-acid rich N-CAM in both neural and glial tissue (Daniloff, J. K. et al. (1986) J. Cell Biol. 103, 929-945). The increased expression of N-CAM after injury and the presence of N-CAM on reactive astrocytes indicates that cell adhesion molecules may play a functional role in regeneration and potential healing after such insults.
Renewed glial cell proliferation and gliosis are important in the response of neural tissue to injury. A reduction in the amount of gliosis after CNS injury may be a significant factor in permitting axonal regeneration across a lesion site. Neuronal regeneration in the adult CNS can occur if regenerating axons are presented with an appropriate environment (Aguayo, A. J. (1985) In Synaptic plasticity, (Cotman, C. W., ed.). Guilford, New York, pp. 457-484). Injury to the CNS of neonatal rats results in less extensive gliosis as compared to the adult (Barrett et al. 1984) possibly facilitating neuronal regeneration. Moreover, gliosis in the adult can be reduced by transplantation of immature astrocytes into the lesion site (Smith, D. B. & Johnson, K. S. (1988) Gene 67, 31-40). Our experiments raise the possibility that this might be due to the high levels of N-CAM expressed by immature astrocytes. Further elucidation of these responses and definition of the signalling pathways that mediate the inhibition of glial cell proliferation facilitate the design of rational therapies to enhance regeneration by CNS neurons.